Antimicrobial Susceptibility Profile and Whole-Genome Analysis of a Strong Biofilm-Forming Bacillus Sp. B87 Strain Isolated from Food

Members of the Bacillus cereus group are considered to be foodborne pathogens commonly associated with diarrheal and emetic gastrointestinal syndromes. Biofilm formation is a major virulence determinant of various pathogenic bacteria, including the B. cereus strains, since it can protect the bacteria against antimicrobial agents and the host immune response. Moreover, a biofilm allows the exchange of genetic material, such as antimicrobial resistance genes, among the different bacterial strains inside the matrix. The aim of the current study was to genotypically and phenotypically characterize Bacillus sp. B87, a strain that was isolated from food and which exhibited strong biofilm-forming capacity. Based on the analysis of the phylogenetic relationship, the isolate was phylogenetically mapped close to Bacillus pacificus. Antimicrobial susceptibility testing revealed that the isolate was resistant to tetracycline and β-lactam antimicrobial agents, which corresponded with the genotypic characterization using the whole-genome analysis. The genome of Bacillus sp. B87 carried the three-component non-hemolytic enterotoxin (NHE), which is a type of enterotoxin that causes diarrheal symptoms. In addition, the genome also contained several genes that participate in biofilm formation, including the pelDEADAFG operon. These findings expand our understanding of antimicrobial resistance and virulence in Bacillus species based on the link between genotypic and phenotypic characterization.


Introduction
Bacillus cereus are Gram-positive, spore-forming bacteria that inhabit food, soil, and other natural media. B. cereus is a known human pathogen that causes food poisoning with emetic or diarrheal symptoms. Emetic strains of B. cereus can secrete the highly thermo-and pH-resistant toxin cereulide, which is pre-formed in food and causes vomiting a few hours after consumption [1]. Cereulide is an ionophoric dodecadepsipeptide that is produced by cereulide synthetase or Ces non-ribosomal peptide synthetase. For diarrheal strains of B. cereus, spores in contaminated food are consumed by the host, germinate within the small intestine, and the resulting vegetative cells then grow and produce enterotoxins. Three types of enterotoxins are secreted by B. cereus: the three-component enterotoxin hemolysin BL (HBL), the three-component non-hemolytic enterotoxin (NHE), and the single-component enterotoxin cytotoxin K (CytK) [2]. In addition to enterotoxins, B. cereus produces several other toxins and degradative enzymes, with most of these products controlled by the PlcR transcriptional activator [3]. PlcR is one of the B. cereus quorumsensing systems that helps the bacterium adapt to diverse conditions [4].
B. cereus is typically resistant to β-lactam antimicrobial agents, such as penicillin G, ampicillin, and cefotaxime [5], due to the production of β-lactamase enzymes [6]. Bacterial resistance to commonly used antimicrobial agents, such as erythromycin, tetracycline, and streptomycin, can be a consequence of both nature and nurture [5,7]. Tetracycline is a broad-spectrum antimicrobial agent with activity against a wide range of bacteria, including Gram-positive and Gram-negative isolates. B. cereus is generally susceptible to tetracycline, but the resistance of B. cereus to this antimicrobial agent has been reported in some countries [8].
Biofilm formation is a major virulence determinant of various pathogenic bacteria, especially in the B. cereus group [9]. The formation of biofilms by bacteria can be associated with chronic infections in human and animal hosts. Moreover, biofilm formation allows the development and transfer of antimicrobial resistance through the bacterial interactions that occur within the biofilm [10,11]. The key genes for biofilm formation comprise those encoding biofilm transcriptional regulators, matrix structural genes, potential extracellular DNA synthesis genes, and cyclic-di-GMP metabolism genes [12]. In addition, several gene loci are involved in biofilm formation, including genes encoding the lipopeptide kurstakin, genes encoding the cyclic-di-GMP responsive effector protein BspA, and genes encoding the c-di-GMP synthesizing enzyme [13][14][15][16]. However, there may be strain-dependent variation in the mechanisms of biofilm formation among members of the B. cereus group.
The accessibility of whole-genome sequencing (WGS) has facilitated the assessment of bacterial genomes through bioinformatics analysis for the genetic potential to produce virulence factors and proteins involved in antimicrobial resistance and biofilm formation. In this study, the strong biofilm-forming strain, Bacillus sp. B87, isolated from food, was characterized genotypically and phenotypically, and genomic comparisons with other relevant B. cereus genomes were performed. The study aimed to generate insights into the genetic basis of antimicrobial resistance and virulence of this foodborne pathogen.

Bacterial Strains and Culture Conditions
The Bacillus sp. B87 used in this study was isolated from a spicy mussel salad in Pathum Thani province, Thailand [17]. Bacteria were aerobically grown in Luria-Bertani (LB) broth (Difco Laboratories, Detroit, MI, USA), with shaking at 180 rpm at 35 ± 2 • C.

Biofilm Formation Assay
The biofilm formation assay was based on a previously described protocol with minor modifications [18]. Briefly, Bacillus sp. B87 was grown overnight in LB broth at 35 • C and 180 rpm to generate inoculum cultures. Overnight cultures were adjusted to an optical density at 600 nm (OD 600 ) of 0.01 in LB. Next, 100 µL of the adjusted bacterial culture was added to each well of a pre-sterilized 96-well flat-bottomed polystyrene microtiter plate, followed by incubation at 35 • C and 50 rpm for 24 h. Planktonic bacteria were then removed, and the wells were washed with distilled water and air-dried. Biofilm cells were stained with 200 µL of 0.3% crystal violet for 10 min, washed with distilled water, and air-dried. The crystal violet in the biofilm cells was solubilized with 200 µL of 70% ethanol, and the optical density at 590 nm (OD 590 ) was measured.

Whole-Genome Sequencing, Assembly, and Annotation
Genomic DNA was extracted from Bacillus sp. B87 using a GF-1 Bacterial DNA Extraction Kit (Vivantis Technologies, Selangor, Malaysia) according to the manufacturer's instructions. DNA quality was assessed via spectrophotometry and gel electrophoresis. Purified high molecular weight DNA samples with a 260/280 nm absorbance ratio of 1.8-2.0 and a 260/230 nm absorbance ratio of 2.0-2.2 were used for library construction and sequencing. The DNA sequencing library was prepared using a QIAGEN FX kit (Qiagen, Valencia, CA, USA), which fragments the gDNA using an enzymatic reaction, cleans the fragmented DNA with magnetic beads, and then ligates an adaptor index to the fragmented DNA. The quality and quantity of the indexed libraries were determined using an Agilent 2100 Bioanalyzer and a Denovix fluorometer, and the libraries were then pooled in equimolar quantities. Cluster generation and paired-end 2 × 150 nucleotide read sequencing were performed on an Illumina HiseqXten (Illumina Inc., San Diego, CA, USA).
The quality of the raw sequencing reads was assessed using FASTQC software. Adaptors and poor-quality reads were removed using Fastp, and the filtered reads were used as inputs for the Unicycler genome assembly program. The genome of Bacillus sp. B87 was annotated with the Rapid Annotation using Subsystem Technology tool kit (RASTtk) in PATRIC (Pathosystems Resource Integration Center). Sequences were queried using the BTyper tool, the Virulence Factor Database (VFDB) and Victors resource (for virulence factors), and the Comprehensive Antibiotic Resistance Database (CARD) and the NCBI National Database of Antibiotic Resistant Organisms (NDARO) (for antimicrobial resistance). In addition, the functional annotation of genes in terms of the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology assignments and predictions of KEGG pathways were performed through the KEGG Automatic Annotation Server (KAAS; https://www.genome.jp/kegg/kaas/ accessed on 18 October 2021) using the bi-directional best hit (BBH) method [21]. The circular genome map was constructed by using the circular viewer of PATRIC. A comparison of syntenic analyses in Bacillus sp. B87 and other species of the genus Bacillus was performed by using the Easyfig program [22].

Phylogenetic Analysis
A phylogenomic tree based on the core genes was generated with PATRIC Phylogenetic Tree Building Service [23]. The default was set for codon trees, which utilizes both the protein and gene sequences from PATRIC's global protein families (PGFams). Protein sequences were aligned using MUSCLE, and the nucleotide coding gene sequences were aligned using the Codon_align function of BioPython. A concatenated alignment of all proteins and nucleotides was converted to a phylip formatted file, and then a partitions file for RaxML was constructed. Support values were created using 100 rounds of the "Rapid" bootstrapping option of RaxML. The phylogenomic classification of Bacillus sp. B87 was also performed using the Type (strain) Genome Server (TYGS), a free bioinformatics platform for a whole-genome-based taxonomic analysis [24].

Nucleotide Sequence Accession Numbers
The draft genomes of Bacillus sp. B87 were deposited in the NCBI database under the accession numbers SRR16129018. The BioProject ID in GenBank is PRJNA7260213.

Biofilm-Forming Ability of Bacillus Sp. B87
The ability of Bacillus sp. B87 to produce biofilm was determined in comparison with B. cereus ATCC 14579. A microtiter plate assay for biofilm formation revealed that the biofilm level of Bacillus sp. B87 (OD 590 0.545 ± 0.149) after incubation for 24 h was significantly higher than that of B. cereus ATCC 14579 (OD 590 0.035 ± 0.002) (Figure 1). matics platform for a whole-genome-based taxonomic analysis [24].

Nucleotide Sequence Accession Numbers
The draft genomes of Bacillus sp. B87 were deposited in the NCBI database under the accession numbers SRR16129018. The BioProject ID in GenBank is PRJNA7260213.

Biofilm-Forming Ability of Bacillus sp. B87
The ability of Bacillus sp. B87 to produce biofilm was determined in comparison with B. cereus ATCC 14579. A microtiter plate assay for biofilm formation revealed that the biofilm level of Bacillus sp. B87 (OD590 0.545 ± 0.149) after incubation for 24 h was significantly higher than that of B. cereus ATCC 14579 (OD590 0.035 ± 0.002) ( Figure 1).

Antimicrobial Resistance Profile of Bacillus sp. B87
Bacillus sp. B87 was tested for susceptibility to 11 selected antimicrobial agents, as shown in Table 1

Antimicrobial Resistance Profile of Bacillus Sp. B87
Bacillus sp. B87 was tested for susceptibility to 11 selected antimicrobial agents, as shown in Table 1

Genetic Features of Bacillus Sp. B87
Genomic features and annotation information for the genome of Bacillus sp. B87 are summarized in Table 2. The draft genome sequence had an estimated length of 5,448,163 bp, a GC content of 35.18%, and contained 5661 coding sequences. The circular representation of the Bacillus sp. B87 draft genome was generated using the circular viewer of PATRIC and is shown in Figure 2. Potential genes in the draft genome of Bacillus sp. B87 were investigated and annotated based on different biological processes and metabolic pathways using the RAST server ( Figure 3). The predicted genes included 711 genes involved in metabolism, 272 genes involved in cellular processes, and 142 genes involved in stress response, defense, and virulence. Genes involved in prophages, transposable elements, and plasmids were also found in the draft genome of Bacillus sp. B87, and were classified as: subclass: pathogenicity islands; subsystem name: Listeria Pathogenicity Island LIPI-1 extended. These pathogenicity islands were also present in the genomes of B. cereus ATCC 14579 and B. anthracis str. Ames.    A phylogenetic tree based on core genes was reconstructed in PATRIC using the whole-genome sequence of Bacillus sp. B87 ( Figure 4A). This phylogenomic analysis revealed that Bacillus sp. B87 was closely related to strains of Bacillus pacificus. The 16S rRNA A phylogenetic tree based on core genes was reconstructed in PATRIC using the wholegenome sequence of Bacillus sp. B87 ( Figure 4A). This phylogenomic analysis revealed that Bacillus sp. B87 was closely related to strains of Bacillus pacificus. The 16S rRNA gene ( Figure 4B) and whole-genome ( Figure 4C) phylogeny reconstructions using TYGS confirmed a close association of Bacillus sp. B87 with B. pacificus.

Antimicrobial Resistance Genes
Antimicrobial resistance (AMR) genes that are associated with resistance to one or more antimicrobial agents were predicted based on the CARD and NDARO databases, and Bacillus sp. B87 contained nine genes connected with resistance to different antimicrobial agents (Table 3). Moreover, 43 AMR genes were annotated according to the PATRIC database using K-mer Search (Supplementary Table S1). These findings might be associated with the observed antimicrobial resistance phenotypes to β-lactam antimicrobial agents and tetracycline shown in Table 1. gene ( Figure 4B) and whole-genome ( Figure 4C) phylogeny reconstructions using TYGS confirmed a close association of Bacillus sp. B87 with B. pacificus.

Biofilm Formation Genes
According to RASTtk, available in PATRIC, the genes involved in the formation of biofilm by Bacillus sp. B87 were identified. Nine genes were detected (Table 4) according to the following hierarchical classification: superclass: cellular process; class: microbial communities; subclass: quorum sensing and biofilm formation. Key biofilm-formation genes were also investigated using the KAAS database, and this analysis identified the genes encoding biofilm transcriptional regulators, matrix protein-encoding genes, putative matrix polysaccharide synthesis genes, and extracellular DNA (eDNA) synthesis genes ( Table 5).

Virulence Factor Genes
Thirteen genes in the draft genome of Bacillus sp. B87 were classified as virulence factors according to the Victors (nine genes) and VFDB (four genes) databases (Table 6). Among these genes were nheA, nheB, and nheC, which encode the three-component NHE complex, a type of enterotoxin that causes diarrheal symptoms. However, the cytotoxin K virulence factor, which is also associated with diarrheal illness and was present in B. cereus ATCC 14579, was not detected in Bacillus sp. B87 (Supplementary Table S2). Virulence genes were also predicted by using the BTyper tool, and this analysis predicted 17 virulence genes in the genome of Bacillus sp. B87 (Table 6).

Discussion
This study aimed to genotypically and phenotypically characterize Bacillus sp. B87, a strain with strong biofilm-forming activity that was previously isolated from a spicy mussel salad in Pathum Thani province, Thailand. The phylogenetic analysis revealed that Bacillus sp. B87 was closely related to B. pacificus, which had been reported to be isolated from the sediment of the Pacific Ocean [25]. Currently, little is known about the phenotype and genotype of this bacterial strain.
The alignment of the Bacillus sp. B87 genome indicated that the isolate possesses the genes nheA, nheB, and nheC, but not hblA, hblC, hblD, or cytK. In B. cereus, the toxins that are associated with diarrheal diseases are HBL, NHE, CytK, and enterotoxin FM [26][27][28][29][30][31][32]. Indeed, isolates that carry the genes encoding HBL might be more virulent [33]. However, a strain lacking the HBL operon-B. cereus ATCC 10987-was reported to exhibit strong cytopathogenic activity in Vero cells, since it could produce a large amount of the NHE mRNA [34]. The genome of Bacillus sp. B87 also contains Listeria Pathogenicity Island LIPI-1 extended. LIP-1 in Listeria monocytogenes harbors several important genes, including prfA, plcA, hly, mpl, actA, and plcB, which are involved in host invasion and cellular proliferation [35]. Orthologs for LIP-1 were also detected in B. cereus ATCC 14579 and B. cereus strain FORC_021, which was isolated from a knife used at a sashimi restaurant in the Republic of Korea [36]. However, the coding sequence (CDS) counts of LIPI-1 genes in each strain were dissimilar in that report. The presence of Listeria Pathogenicity Island LIPI-1 extended in Bacillus sp. B87 might be responsible for the virulence of this strain and requires further investigation.
B. cereus is typically resistant to β-lactam antimicrobial agents, including penicillin G, ampicillin, and amoxicillin-clavulanic acid [17,20,37,38]. Some bacterial strains can produce β-lactamase enzymes that are responsible for the resistance to β-lactam antimicrobial agents. Three different β-lactamases have been classified in strains of B. cereus, namely, β-lactamase I, II, and III [39]. B. cereus β-lactamase II (BcII) is a heat-stable metallo-β-lactamase (MBL) that shares high sequence homology with Bla2 from B. anthracis [40]. MBL catalyzes the hydrolysis of β-lactam antimicrobial agents, including penicillin, cephalosporin, carbapenem, and cephamycin [41]. However, BcII catalyzes the hydrolysis of penicillin at higher rates than cephalosporin and carbapenem [42]. The current study revealed the presence of the BcII gene in the draft genome of Bacillus sp. B87, and this gene might play an important role in the β-lactam resistance phenotype of the isolate.
Bacillus sp. B87 carries the tet(45) gene, which contributes to the tetracycline resistance phenotype in this isolate. Tet45 is a tetracycline efflux pump closely related to TetL [43]. Homologs of Tet45 have been found in the genomes of strains of B. cereus and B. thuringiensis. Furthermore, tetA-carrying B. cereus was reported to be susceptible to tetracycline [44]. Moreover, some isolates of B. cereus were phenotypically resistant to tetracycline, even though they did not carry tetA, tetB, or tetC, and this might be due to the presence of other tetracycline resistance genes, such as tetM and tetL [45]. B. cereus is generally susceptible to tetracycline, but the resistance of B. cereus to tetracycline has been reported in some countries [8]. In the current study, Bacillus sp. B87 was resistant to tetracycline, since it possessed the gene encoding the tetracycline resistance efflux pump. This phenotype is concordant with a previous report on B. cereus strain MS532a, which presented a tetracyclineresistant phenotype and carried the tet(45) tetracycline resistance gene [38]. Horizontal gene transfer may have contributed to the dissemination and persistence of tet (45) in environments such as a poultry litter-impacted soil [43]. The presence of the tet(45) gene in the genome of Bacillus sp. B87, a strain isolated from spicy mussel salad, may be due to the contamination of vegetables, mussels, or other ingredients. Tetracycline-resistant isolates of B. cereus have previously been shown to carry the tet(L) gene on a plasmid, while other species of the genus Bacillus carry either tet(L) or tet(K) on plasmids and/or in the chromosome [46][47][48]. Both genes-tet(L) and tet(K)-can occasionally be mobilized in the presence of conjugative plasmids, but are not themselves able to independently transfer, thus decelerating their spread within the population [45]. The ability to form biofilm in bacteria is an important mechanism that allows the bacteria to resist antimicrobial agents and disinfectants, as well as to evade the host immune system. B. cereus can form biofilms on various surfaces [49][50][51], including plastic, glass wool, and stainless steel, and the biofilm cells were more resistant to sanitizers compared with planktonic cells [52]. Biofilm plays an important role in the exchange of antimicrobial resistance genes [53]. In strains of Escherichia coli and Pseudomonas aeruginosa, sub-inhibitory concentrations of tetracycline and cephradine induce biofilm formation and increase the transfer rate of the pB10 plasmid among the biofilm biomass at higher rates compared with no antimicrobial treatment [54].
The findings from the current study were congruent with a previous report [55] that resistant bacteria could form biofilms. Tetracycline-resistant Bacillus sp. B87, with strong biofilm-formation ability, may develop greater resistance to different antimicrobial agents and other environmental stressors if left unmonitored.
The regulation network controlling B. cereus biofilm formation involves several pathways. PlcR is a pleiotropic regulator that controls the expression of genes encoding several enterotoxins, hemolysins, phospholipases, and proteases in B. cereus [9,56]. The plcR gene is instrumental in biofilm formation, since the deletion of this gene in B. cereus ATCC 14579 resulted in a significant increase in the amount of biofilm [57]. Two roles have been reported for the regulator CodY: the repression of biofilm formation in B. cereus ATCC 14579 [58] and the promotion of biofilm formation in B. cereus UW101C [59]. The codY gene operon has a role in pellicle biofilm formation and swarming motility [60]. An operon including tasA, tapA, and sipW has been proposed to be involved in biofilm formation in B. subtilis [61]. The transcription of these genes is regulated by SinI and repressed by SinR [62]. The transcription of sinI is activated by the master regulator of sporulation Spo0A [63]. In the present study, Bacillus sp. B87 was found to carry the genes tasA and sipW, which may be sufficient for biofilm production. The deletion of the genomic region encoding two orthologs of the amyloid-like protein TasA and SipW signal peptidase inhibited biofilm assembly [64]. However, the genes calY and tapA were not detected in the isolate. Mutations in tasA or calY did not completely prevent biofilm formation. Moreover, the lack of tapA may not completely interrupt biofilm production, which was congruent with a previous report [65].
Several gene loci are involved in biofilm formation, and there may be strain-to-strain variability in matrix component utilization among isolates of B. cereus. For example, the pelDEA DA FG operon identified in strain ATCC 10987 is a crucial locus for biofilm formation in B. cereus. However, this locus is not present in strain ATCC 14579 of this species. This operon might be required for biofilm formation in Bacillus cereus strains, at least on polystyrene surfaces, since ATCC 10987 formed biofilms, while ATCC 14579 did not form biofilms on polystyrene 96-well plates [66]. In addition, the deletion of any of pelDEA DA FG in ATCC 10987 resulted in the reduction of bacterial adhesion to the wells of the plastic microtiter dish [67]. The bioinformatics analysis and validation of Pel production in Bacillus sp. B87 in the current study suggested that the pelDEA DA FG operon was present and potentially functional in biofilm-forming strains. In addition, the presence of the gene recA in the genome of Bacillus sp. B87 may enhance the biofilm-forming capacity of this isolate, since it has been reported that the biofilm formation and swarming motility of B. cereus 905 are promoted by RecA [68]. However, further investigation via a gene deletion approach is required to confirm this theory.

Conclusions
This study reported the phenotypic and genotypic characterization of Bacillus sp. B87, an isolate with strong biofilm-forming capacity. The isolate was resistant to β-lactam antimicrobial agents and tetracycline, since it carried BcII and tet (45) genes in the genome. Consequently, the strain can survive in tetracycline-containing environments where it may induce the biofilm-forming capacity of this isolate, and this assumption needs to be further elucidated. Moreover, the presence of the pelDEA DA FG operon in the genome of Bacillus sp. B87 might play an important role in the biofilm-forming capacity of this isolate. Biofilm formation would allow the bacteria to exchange genetic material among strains, and could potentially lead to the development of greater resistance to different antimicrobial agents and other environmental stressors. In conclusion, the identification of genes encoding virulence factors and antimicrobial resistance in foodborne bacteria should be considered to be potential key points to assess human health risks from the bacteria. Moreover, the findings from this study suggest that WGS analysis could be an effective tool to elucidate the pathogenic potential of the B. cereus group. However, the molecular basis proposed in this study needs to be further clarified through gene knockout and protein characterization, since several gene homologs in members of the B. cereus group have unique and varied functions.
Author Contributions: Conceptualization, P.S. and K.S.; methodology, P.S., R.T. and P.P.; software, K.S.; validation, P.S., K.S. and S.W.; formal analysis, P.S.; writing-original draft preparation, P.S.; writing-review and editing, P.S., K.S., P.P., R.T. and S.W.; visualization, P.S.; supervision, K.S.; project administration, P.S.; funding acquisition, P.S. All authors have read and agreed to the published version of the manuscript. Data Availability Statement: The raw sequence generated for this study was deposited into the National Centre for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under the accession numbers SRR16129018, BioProject ID: PRJNA726021. All the other data supporting the finding of this study are available in this published article and its supplementary information files.